Phytochrome B Represses Teosinte Branched1 Expression and Induces Sorghum Axillary Bud Outgrowth in Response to Light Signals1

Tesfamichael H. Kebrom, Byron L. Burson, and Scott A. Finlayson* Department of Soil and Crop Sciences (T.H.K., S.A.F.), and United States Department of Agriculture Agricultural Research Service (B.L.B.), Texas A&M University, College Station, Texas 77843–2474

Light is one of the environmental signals that regulate the development of shoot architecture. Molecular mechanisms regulating shoot branching by light signals have not been investigated in detail. Analyses of light signaling mutants defective in branching provide insight into the molecular events associated with the phenomenon. It is well documented that phytochrome B (phyB) mutant plants display constitutive shade avoidance responses, including increased plant height and enhanced apical dominance. We investigated the phyB-1 mutant sorghum (Sorghum bicolor) and analyzed the expression of the sorghum Teosinte Branched1 gene (SbTB1), which encodes a putative transcription factor that suppresses bud outgrowth, and the sorghum dormancy-associated gene (SbDRM1), a marker of bud dormancy. Buds are formed in the leaf axils of phyB-1; however, they enter into dormancy soon after their formation. The dormant state of phyB-1 buds is confirmed by the high level of expression of the SbDRM1 gene. The level of SbTB1 mRNA is higher in the buds of phyB-1 compared to wild type, suggesting that phyB mediates the growth of axillary shoots in response to light signals in part by regulating the mRNA abundance of SbTB1. These results are confirmed by growing wild-type seedlings with supplemental far-red light that induces shade avoidance responses. We hypothesize that active phyB (Pfr) suppresses the expression of the SbTB1 gene, thereby inducing bud outgrowth, whereas environmental conditions that inactivate phyB allow increased expression of SbTB1, thereby suppressing bud outgrowth.

The highly ordered arrangement of leaves and in the axil of a leaf to form a bud (for review, see Ward branches and the final shoot architecture are associated and Leyser, 2004; McSteen and Leyser, 2005). Then, de- with a plant’s developmental strategy to ensure its pending on internal and environmental signals, the bud survival and productivity under continuously changing may continue growing to form an axillary shoot or enter growing conditions. A complex developmental pro- into dormancy. Different approaches have been used to gram that integrates genetic mechanisms, physiological study the regulation of axillary shoot development. processes, and environmental signals controls the over- Early decapitation studies and application of plant all form of the plant. The shoot architecture of crop hormones showed that auxin produced in the shoot plants has been modified during their domestication apex inhibits the outgrowth of axillary buds (Thimann and improvement. One of the great achievements in the and Skoog, 1933). Further research indicated that other history of crop improvement, ‘‘The Green Revolution,’’ plant hormones, such as cytokinins and abscisic acid, was due to dwarfing of wheat (Triticum aestivum)and are also involved in regulating branching (for review, other crops to increase nitrogen-use efficiency and re- see Stafstrom, 2000; Shimizu and Mori, 2001). duce lodging. A more complete understanding of the Molecular and genetic approaches have been used developmental programs that control shoot architec- to study the mechanisms of action of plant hormones ture will help to further improve resource-use effi- and to identify genes involved in regulating branch- ciency and yield of crop plants. ing. Genes that control axillary meristem initiation Shoot architecture is largely determined by the num- have been identified in various species (Schumacher ber of axillary shoots produced. Axillary shoot devel- et al., 1999; Greb et al., 2003; Li et al., 2003). Studies opment begins with the initiation of an axillary meristem using branching mutants of Arabidopsis (Arabidopsis thaliana), pea (Pisum sativum), and petunia (Petunia hybrida) are revealing the mechanisms of action of 1 This work was supported by the Texas Agricultural Experiment complex signaling networks of plant hormones that Station. regulate shoot branching (Leyser et al., 1993; Stirnberg * Corresponding author; e-mail sfi[email protected]; fax 979– et al., 1999; Booker et al., 2005; Foo et al., 2005; 845–0456. Snowden et al., 2005). Investigations using these mu- The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy tants suggest the presence of a novel, as yet uniden- described in the Instructions for Authors (www.plantphysiol.org) is: tified, plant hormone-like signal that integrates Scott A. Finlayson (sfi[email protected]). hormonal action during branching. Article, publication date, and citation information can be found at A genetic analysis of the morphological differences www.plantphysiol.org/cgi/doi/10.1104/pp.105.074856. between maize (Zea mays subsp. mays) and its wild

Plant Physiology, March 2006, Vol. 140, pp. 1109–1117, www.plantphysiol.org Ó 2006 American Society of Plant Biologists 1109 Kebrom et al.

developmental processes, including branching in sor- ghum (Sorghum bicolor) and Arabidopsis (Childs et al., 1992, 1997; Reed et al., 1993). We used the phyB null mutant sorghum (phyB-1)as a model for studying the regulation of branching by light. The strong apical dominance of phyB-1, and the early formation and ease of excising axillary buds make sorghum useful for studying the role of light in axillary shoot development. We asked whether light signals perceived by phyB control shoot branching by regulating the expression of branching genes previ- Figure 1. Median longitudinal sections of 4-d-old sorghum shoots. Arrows indicate buds in the axil of the first leaves of phyB-1 (A) and ously identified in other species. We characterized wild-type (B) sorghum seedlings. the enhanced apical dominance in phyB-1 sorghum and investigated the expression of branching-related genes, including the sorghum homologs of the TB1 ancestor teosinte (Zea mays subsp. parviglumis) led to (SbTB1) and dormancy-associated (SbDRM1) genes in the cloning of the Teosinte Branched1 (TB1)gene(Doebley phyB-1 and wild-type axillary buds. In this article, we et al., 1997). The TB1 gene encodes a putative basic report the regulation of expression of the sorghum helix-loop-helix transcription factor that is involved SbTB1 and SbDRM1 genes by light signals perceived in suppressing bud outgrowth (Doebley et al., 1997; by phyB, and phyB’s association with dormancy and Hubbard et al., 2002). An orthologous gene to maize outgrowth of axillary buds. TB1 was cloned from rice (Oryza sativa; OsTB1) and functions in a manner similar to the maize TB1 (Takeda et al., 2003). In both maize/teosinte and rice, RESULTS TB1 is expressed predominantly in the young axillary bud. Further studies of the functional properties of Enhanced Apical Dominance in phyB-1 Mutant Sorghum TB1 will provide insights regarding the regulation of shoot branching at the molecular level. The phyB-1 mutant sorghum fails to produce Several genes that are specifically up-regulated or branches during vegetative development, whereas down-regulated during branching have been identi- the near-isogenic wild-type plants branch profusely. fied. The dormancy-associated genes of pea, PsDRM1 The branching deficiency in phyB-1 could theoretically (Stafstrom et al., 1998b) and PsAD1 (Madoka and arise from either a defect in axillary meristem initia- Mori, 2000), are among the genes identified in cDNA tion or bud outgrowth. We found that the defect occurs libraries from dormant axillary buds. The mRNA abundance of PsDRM1 and PsAD1 in axillary buds declines after decapitation. However, while the ex- pression of these genes correlates strongly with bud dormancy, their functions are not known. It is well established that light quality (red:far red [R:FR]) is one of the environmental signals that regu- late shoot branching (Casal et al., 1986; Wan and Sosebee, 1998). However, little information exists re- garding the molecular mechanisms regulating shoot branching in response to light. Light signals may interact with plant hormones to regulate plant growth and development (Halliday and Fankhauser, 2003). Recent findings indicate that light and auxin interact in the regulation of adventitious root formation in Arabidopsis (Sorin et al., 2005). To fully understand the regulation of axillary shoot development, the in- teraction of light and hormonal and other environ- mental signals should be investigated. Plants use light signals perceived by photoreceptors to coordinate all stages of growth and development from germination to flowering. The phytochrome family of photoreceptors is involved in deetiolation, vegetative development, and flowering time in both dicots and monocots (Mathews and Sharrock, 1996; Figure 2. Bud length (A) and seedling height (B) of phyB-1 and wild- Sawers et al., 2005). Mutation in one of the family type sorghum. Inset shows bud length at 7 DAP, 8 DAP, and 9 DAP (not members, PHYTOCHROME B (PHYB), affects several visible in the main figure). Data are mean 6 SE; n 5 10.

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expressed in mature stems and roots. The Arabidopsis gene orthologous to PsDRM1, AtDRM1,hassimilar expression patterns to PsDRM1 (Stafstrom et al., 1998a; Tatematsu et al., 2005). A BLAST search (TBLASTN; Altschul et al., 1991) identified a sorghum expressed sequence tag (accession no. CN135114, hereafter called Figure 3. SbTB1 mRNA abundance in leaf, sheath, root, and bud of SbDRM1) encoding the entire SbDRM1 open reading sorghum seedlings at 9 DAP. The sorghum ubiquitin gene (SbUBQ)was frame with a deduced amino acid sequence 59% and 53% used as a loading control. identical to the PsDRM1 (accession no. AAB84193.1) and AtDRM1 (accession no. AAC26202) proteins, re- spectively (Fig. 4). The amino acid sequence iden- in bud outgrowth since equivalent buds are formed tity between PsDRM1 and AtDRM1 is 66% (Stafstrom early in the development in both phyB-1 and wild-type et al., 1998a). Northern-blot analysis showed that the sorghum (Fig. 1). The buds in the axil of the first leaf of SbDRM1 mRNA is about 1.15 kb in length. SbDRM1 is both phyB-1 and the wild type grow at the same rate expressed in leaves, sheaths, and roots of sorghum until 7 d after planting (DAP). Then they begin to show seedlings and at low levels in outgrowing buds (Fig. 5). different developmental fates (Fig. 2A). While the buds of the wild type continue elongation, bud outgrowth is Abundance of SbTB1 and SbDRM1 mRNA in phyB-1 inhibited in phyB-1. Axillary buds are formed at all and Wild-Type Axillary Buds nodes of the main shoot of phyB-1 (data not shown). These buds remain dormant and branching is observed The expression of the SbTB1 and SbDRM1 genes in only when the main shoot begins flowering. The phyB-1 sorghum axillary buds was correlated with the dor- seedlings were taller than the wild type, showing mant state of the buds. At 7 DAP and 9 DAP, SbTB1 enhanced growth of the main shoot as a result of mRNA was detected in the axillary buds of both constitutive shade avoidance (Fig. 2B). phyB-1 and wild-type seedlings (Fig. 6A). However, SbTB1 abundance was more than 2-fold higher in the Expression of SbTB1 in Different Organs of phyB-1 buds compared to the level in the wild type Sorghum Seedlings (Fig. 6B). The expression pattern of SbDRM1 was different from that of SbTB1 mRNA. At 7 DAP, when SbTB1 (accession no. AF322132) exists as a single their size was comparable (Fig. 2), SbDRM1 message is copy in the sorghum genome with 93.9% nucleotide hardly detected in the buds of phyB-1 and the wild identity with the maize TB1 gene (Lukens and Doebley, type (Fig. 6A). At 9 DAP, when the buds of the wild 2001). The maize TB1 mRNA is 1.5 kb in size and type were rapidly elongating while those of phyB-1 accumulates in husks, axillary inflorescence primor- were suppressed, the mRNA level of SbDRM1 was dia, and axillary meristems (Doebley et al., 1997; more than 5-fold higher in the buds of phyB-1 than in Hubbard et al., 2002). OsTB1 is expressed in the basal the wild type (Fig. 6C). part of the shoot apical meristem, in vascular tissue in the pith, and in the entire axillary bud (Takeda et al., SbTB1 and SbDRM1 mRNA Abundance in Axillary 2003). We investigated the abundance of SbTB1 in Buds of Wild-Type Seedlings Grown at High different parts of sorghum seedlings. The SbTB1 and Low Densities mRNA of about 1.7 kb in size was detected only in the buds (Fig. 3). The enhanced apical dominance of phyB-1 sorghum is consistent with a constitutive shade avoidance re- Expression of SbDRM1 in Different Organs of sponse. Shade avoidance responses are also observed Sorghum Seedlings in the natural environment when wild-type plants are grown at high density that lowers the R:FR. In an PsDRM1 is expressed in dormant axillary buds, and attempt to simulate the constitutive shade avoidance its expression is suppressed by decapitation and auxin response of phyB-1 sorghum in the wild type, wild-type (Stafstrom et al., 1998b; Stafstrom, 2000). PsDRM1 is also seedlings were grown at high (3,000 seedlings m22) and

Figure 4. Sequence similarity among sorghum, pea, and Arabidopsis dormancy-associated proteins (SbDRM1, PsDRM1, and AtDRM1, respectively).

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DISCUSSION We investigated the regulation of branching by light signals perceived by phyB in the branching-deficient phyB-1 null mutant sorghum. Mutants used previously to study the molecular mechanisms of branching are defective either in initiation of axillary meristems or Figure 5. SbDRM1 mRNA abundance in leaf, sheath, root, and bud of outgrowth of buds (Napoli et al., 1999; Ward and sorghum seedlings at 9 DAP. The sorghum ubiquitin gene (SbUBQ)was Leyser, 2004). Our results show that the initiation of used as a loading control. axillary meristems and formation of buds occur nor- mally in phyB-1; however, the outgrowth of those buds was inhibited, indicating that the role of light signals low (300 seedling m22) plant densities. In those seed- in branching is limited to bud dormancy and outgrowth. lings grown at a high density, axillary bud outgrowth Bud outgrowth was also inhibited in wild-type plants was suppressed at about 9 DAP (Fig. 7A). While high grown at high plant density or with supplemental FR density affected axillary bud elongation, the height of light, demonstrating the regulation of axillary buds by seedlings at both planting densities was the same environmental signals soon after their formation. during the measurement period (Fig. 7B). Supplemental FR light treatment of wild-type seed- SbTB1 mRNA accumulation in the buds of wild- lings, started at 7 DAP, inhibited the outgrowth of type seedlings grown at both plant densities decreased buds in the first leaf axil of all treated seedlings. over time (Fig. 8). The abundance of SbTB1 mRNA was However, when FR treatment was started at 9 DAP, the higher in the axillary buds from high plant density compared to low plant density at both 7 DAP and 9 DAP (Fig. 8). At 7 DAP, SbDRM1 mRNA abundance was only 2.2-fold higher in high density compared to low density (Fig. 9). However, relative to wild type at 7 DAP, the level of SbDRM1 mRNA at 9 DAP was increased to 18.6- and 3.7-fold at high and low plant densities, respectively. At 9 DAP, SbDRM1 abundance was therefore 5-fold higher in buds from high com- pared to low planting density.

SbTB1 and SbDRM1 mRNA Abundance in Axillary Buds of Wild Type Grown with and without Supplemental FR Low R:FR reduces the proportion of the active form of phyB (Pfr), thereby triggering the shade avoidance response. Wild-type seedlings were grown until 7 DAP, when bud length in the axil of the first leaf and seedling height were measured (control, 7 DAP). Then, starting at 7 DAP and continuing for the next 2 d, some of the seedlings were irradiated with FR light from the sides (FR, 9 DAP), while others con- tinued growth without supplemental FR light (con- trol, 9 DAP). The supplemental FR light treatment suppressed the outgrowth of buds in the axil of the first leaf (Fig. 10A) and increased seedling height (Fig. 10B). The abundance of SbTB1 and SbDRM1 mRNAs reflects the enhanced apical dominance induced by low R:FR (Fig. 11A). Compared to the abundance in the control at 7 DAP, the SbTB1 mRNA abundance at 9 DAP was slightly reduced in the control, whereas it was increased in the FR-treated seedlings. The mRNA abundance of SbTB1 was 2.8-fold higher in the FR- Figure 6. A, SbTB1 and SbDRM1 mRNA abundance in the buds from treated seedlings compared to the control at 9 DAP the first leaf axils of phyB-1 and wild-type sorghum seedlings. B and C, (Fig. 11B). The SbDRM1 mRNA level was almost 18- Relative quantitation of SbTB1 and SbDRM1 mRNA levels, respec- fold higher in axillary buds of seedlings treated with tively. The sorghum ubiquitin gene (SbUBQ) was used as a loading FR compared to control (Fig. 11B). control.

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The pattern of SbDRM1 accumulation agrees with that observed in pea and Arabidopsis, implying that conserved mechanisms of dormancy and outgrowth may operate in both monocots and dicots—an obser- vation also supported by the orthologous function of MAX-related genes in rice (Ishikawa et al., 2005). Since auxin represses PsDRM1 in pea (Stafstrom, 2000), the suppression of SbDRM1 mRNA accumulation by light suggests that similar dormancy mechanisms are in- duced by both auxin and light. Phytochrome regula- tion of gene expression has been shown to be both auxin dependent and auxin independent (Tanaka et al., 2002). Whether the suppression of SbDRM1 mRNA by light is auxin dependent or auxin independent needs to be investigated. It is not known whether decapitation- induced and non-decapitation-induced bud outgrowth (i.e. via light) are regulated through the same pathway (Napoli et al., 1999). Further studies on the regulation of expression of SbDRM1 mRNA and related genes by Figure 7. Bud length (A) and seedling height (B) of wild-type sorghum light signals will help resolve such questions. seedlings grown at high and low plant densities. Data are mean 6 SE; The TB1 gene was identified as one of five quanti- n 5 9 or 10. tative trait loci that distinguish the morphology of maize from its wild ancestor, teosinte (Doebley and Stec, 1991). Compared to teosinte, maize exhibits en- buds in the first leaf axil of some seedlings were hanced apical dominance. Sequence comparison of the arrested, while in others they escaped the inhibitory maize and teosinte TB1 alleles indicated no difference signal and elongated (data not shown). It is noteworthy in the predicted coding region of the two alleles (Doebley that all the buds in the axil of the second leaves of et al., 1997); however, the expression of the maize TB1 those plants treated with FR at 9 DAP were arrested was twice that of teosinte, suggesting differences in (data not shown). The inconsistency in the response of theregulationofexpressionofthegene(Doebleyetal., buds in the axil of the first leaf to delayed FR light treatment was observed in repeated experiments. The ‘‘escape’’ phenomenon may be similar to the regula- tion of bud dormancy and outgrowth by auxin (for review, see Cline, 1997), in which decapitation- induced bud outgrowth can be suppressed by exoge- nous auxin early after decapitation but not later. These results indicate the presence of a developmental win- dow at which FR light and auxin can inhibit bud outgrowth. The data raise the question of whether the effect of light on bud dormancy and outgrowth is through auxin, or whether both light and auxin act on a common target that regulates the process. Previous research has indicated that DRM1 expres- sion correlates with bud dormancy, and we have used it here as an indicator of a bud’s physiological status. The inhibition of bud outgrowth in phyB-1 sorghum is reflected in the high level of SbDRM1 mRNA in these axillary buds (Fig. 6). The results are confirmed by the high level of SbDRM1 mRNA in the arrested buds of wild type grown at high plant density or with supple- mental FR (Figs. 9 and 11). There was a small increase in SbDRM1 mRNA at 9 DAP compared to 7 DAP in wild type grown under standard conditions (Fig. 6) and in wild type grown at low densities (Fig. 9). These results may seem contradictory to the typical cor- Figure 8. A, Abundance of SbTB1 mRNA in axillary buds in the axil of relation of DRM1 expression with dormancy but the first leaves of wild-type seedlings grown at high and low densities at may indicate an increase in the proportion of nondi- 7 DAP and 9 DAP. B, SbTB1 mRNA quantitation relative to buds of viding cells in the rapidly elongating buds of the wild seedlings grown at low density at 7 DAP. The sorghum ubiquitin gene type. (SbUBQ) was used as a loading control.

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induced an increase in seedling height, which is one of the typical shade avoidance responses. The pattern of accumulation of SbDRM1 mRNA at 9 DAP at high planting density and in the FR-treated seedlings was similar in each case, indicating both treatments in- hibited bud outgrowth through similar downstream mechanisms. However, the level of SbTB1 mRNA at 9 DAP at high planting density was lower than at 7 DAP at both high and low planting densities (Fig. 8), while FR light treatment increased the level of SbTB1 mRNA at 9 DAP compared to the level at 7 DAP (Fig. 11). Takeda et al. (2003) reported that the branching habit of the fine culm 1 mutant rice, which contains a loss-of- function mutation of OsTB1, responds to planting density. They suggested that the regulation of branch- ing in rice in response to density is independent of OsTB1 but dependent on other factors. Since high density did not affect plant height and the effect on SbTB1 expression is relatively small, it is unlikely that suppression of bud outgrowth in our experiment is mainly the result of a phyB-mediated shade avoidance response. In addition to changes in light quality, other undetermined density-derived cues may also inhibit Figure 9. A, Abundance of SbDRM1 mRNA in axillary buds in the axil bud outgrowth at high planting density. of the first leaves of wild-type seedlings grown at high and low densities SbTB1 mRNA is highest in the younger wild-type at 7 DAP and 9 DAP. B, SbDRM1 mRNA quantitation relative to buds of buds (7 DAP) and decreases with time, suggesting that seedlings grown at low density at 7 DAP. The sorghum ubiquitin gene early in development these buds acquire a signal that (SbUBQ) was used as a loading control. inhibits their outgrowth. The fate of these buds, whether to continue growth or enter into dormancy, is determined at a later stage depending on the per- 1997). Increased expression of the TB1 gene in axillary ception of light and possibly other signals required for organs was correlated with suppression of growth their elongation. The results imply that the absence of of those organs (Doebley et al., 1997; Hubbard et al., active phyB to suppress SbTB1 accumulation in the 2002). Increased expression of OsTB1 was also found to phyB-1 axillary buds leads to dormancy, while the suppress axillary bud outgrowth but not their formation suppression of SbTB1 accumulation in the wild-type (Takeda et al., 2003). The elevated SbTB1 mRNA abun- axillary buds by active phyB leads to bud outgrowth. dance in phyB-1 axillary buds compared to wild type and in wild type grown with low R:FR suggests that SbTB1 gene expression is regulated by light signals perceived by phyB. Therefore, phyB may exert its influence on branching in part through modulation of SbTB1 mRNA levels. Branching in maize is relatively insensitive to plant- ing density; whereas branching in teosinte is reduced at high plant density and increases at low plant density. Doebley et al. (1995) suggested that TB1 expression in maize is decoupled from regulation by environmental signals and is constitutively expressed, while teosinte TB1 is expressed under unfavorable growing conditions and repressed under favorable growing conditions. Our results suggest that the reg- ulation of TB1 expression in wild-type sorghum more closely resembles that proposed for teosinte, while the expression in phyB-1 sorghum more closely resembles that proposed for maize. It should be noted that the response of wild-type sorghum seedlings to high planting density was not the same as their response to low FR light treatment during the experimental period. Although bud elon- Figure 10. Effect of supplemental FR light on bud outgrowth (A) and gation was inhibited in both treatments, only FR seedling height (B) of wild-type sorghum. Data are mean 6 SE; n 5 7.

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et al., 1999). Previous studies indicated that the TCP proteins stimulate or repress the growth of an organ in which they are expressed by regulating the expression of several cell cycle-related genes (Kosugi and Ohashi, 1997, 2002; Gaudin et al., 2000; Tremousaygue et al., 2003; Li et al., 2005). Since TB1 represses the growth of organs in which it is expressed, it may directly or indirectly repress growth by interfering with the pro- gression of cell division. We are investigating the expression of several cell cycle genes, such as PCNA, ribosomal protein genes, Cyclins, and CDKs, in the phyB-1 and wild-type sorghum axillary buds to eluci- date the regulation of bud outgrowth by TB1 in re- sponse to light. It is not yet understood whether hormonal signals interact with TB1 to suppress bud outgrowth (McSteen and Leyser, 2005). We have identified several sorghum expressed sequence tags with homology to key genes involved in regulating hormonal signals during axil- lary shoot development, including MAX related (Booker et al., 2005) and AXR1 (Leyser et al., 1993; Stirnberg et al., 1999), and we are investigating their patterns of expression in the sorghum system to un- derstand the complex signaling networks involved in Figure 11. A, Abundance of SbTB1 and SbDRM1 mRNA in axillary regulating axillary shoot development. buds of wild-type seedlings grown with or without supplemental FR Tatematsu et al. (2005) identified cis-elements that light. B, Relative quantitation of SbTB1 and SbDRM1 mRNA levels. The regulate the expression of genes involved in axillary sorghum ubiquitin gene (SbUBQ) was used as a loading control. bud outgrowth in Arabidopsis by analyzing the tran- scriptome of axillary shoots collected from all posi- tions. Since axillary shoots at different phyllotactic Transgenic potato (Solanum tuberosum) plants over- positions are at different developmental stages, it is expressing phyB produced more branches at high difficult to delineate the role of those elements at the plant density (Boccalandro et al., 2003). These results different stages. Using both the monocots such as are consistent with the hypothesis that active phyB sorghum where working with buds comparable in size suppresses accumulation of the TB1 mRNA, thereby and at similar positions is easier and dicots such as promoting bud outgrowth. Recently, a potato gene pea and Arabidopsis where decapitation, grafting, and (sttcp1) with a function similar to that of the maize TB1 genetic manipulation are easier, a more complete gene was cloned (Faivre-Rampant et al., 2004). It understanding of the regulation of plant architecture would be interesting to study the level of expression may be achieved. of the sttcp1 gene in the phyB-overexpressing and wild- type potato. Devlin et al. (2003) studied genome-wide gene ex- MATERIALS AND METHODS pression changes in phyB mutant, phyA phyB double mutant, and wild-type Arabidopsis in response to Plant Materials and Growing Conditions shade and identified 301 genes that demonstrated Wild-type and phyB-1 mutant sorghum (Sorghum bicolor) seedlings were altered expression. Some of these genes encode pro- grown in a growth chamber with incandescent and fluorescent lamps, main- teins that are known to function in light and hormone tained at 31°C/22°C day/night temperatures with a 12-h photoperiod. The 2 2 signaling, transcription regulation, and protein degra- photosynthetically active radiation was approximately 600 mmol m 2 s 1 and the R:FR 3.0. Seeds were sown at a rate of 300 m22 on trays containing 7-cm- dation. In some developmental programs, phytochrome deep cells filled with growth medium prepared as described by Beall et al. action involves a direct interaction with transcription (1991). To study the effect of plant density, seeds were sown at a rate of 300 and factors to regulate the expression of light-responsive 3,000 m22 for the low and the high plant density, respectively. Seedlings near the genes. Phytochromes may also act through posttransla- perimeter of the trays were avoided during sampling. To study the effect of tional regulation of the level of transcription factors by shade signals (R:FR) on bud outgrowth, seedlings were grown in 50-mL tubes, one seedling per tube, and the tubes were supported on a rack arranged at a directed proteolysis to indirectly regulate gene expres- seedling density of 900 m22. At this rate, shading due to density was not sion (Quail, 2002). The mechanism by which phyB observed as detected by measurement of bud elongation until 10 DAP (data not regulates SbTB1 abundance remains to be discovered. shown). At 7 DAP, uniform seedlings were selected, divided into two groups, 2 The TB1 gene belongs to the TCP family of tran- arranged on a rack at a seedling density of 300 seedlings m 2, and then one group was treated with supplemental FR (FR, 9 DAP) while the other group scription factors with a noncanonical basic helix- was grown as control (control, 9 DAP). Supplemental FR was applied using FR loop-helix domain that is predicted to function in light-emitting diode arrays directed from the sides starting at 7 DAP. FR-treated DNA-binding and protein-protein interactions (Cubas and control seedlings were grown in the same growth chamber under the same

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conditions except that the FR-treated seedlings had additional FR light from Beall FD, Morgan PW, Mander LN, Miller FR, Babb KH (1991) Genetic R the sides. regulation of development in Sorghum bicolor:V.Thema3 allele results in gibberellin enrichment. Plant Physiol 95: 116–125 Boccalandro HE, Ploschuk EL, Yanovsky MJ, Sanchez RA, Gatz C, Casal Histological Studies JJ (2003) Increased phytochrome B alleviates density effects on tuber yield of field potato crops. Plant Physiol 133: 1539–1546 Young elongating shoots of phyB-1 and the wild type were collected about Booker J, Sieberer T, Wright W, Williamson L, Willett B, Stirnberg P, 5 mm from the base of seedlings and fixed in FAA (70% ethanol, 37% Turnbull C, Srinivasan M, Goddard P, Leyser O (2005) MAX1 encodes a formaldehyde-acetic acid, 18:1:1) for at least 24 h, and then stored in 70% cytochrome P450 family member that acts downstream of MAX3/MAX4 ethanol. The young stems were dehydrated in a tertiary butyl alcohol series, to produce a carotenoid-derived branch-inhibiting hormone. Dev Cell 8: embedded in Tissueprep (Fisher Scientific), sectioned at 15 mm with a rotary microtome, and placed on microscope slides that were kept at 40°Cto50°C for 443–449 Casal JJ, Sanchez RA, Deregibus VA at least 24 h. The slides were then stained in Safranin-O fast-green series using (1986) The effect of plant density on an HMS series programmable slide stainer (Carl Zeiss). Two drops of tillering: the involvement of R/FR ratio and the proportion of radiation Permount mounting medium (Fisher Scientific) were placed on each slide intercepted per plant. Environ Exp Bot 26: 365–371 Childs KL, Cordonnier-Pratt M-M, Pratt LH, Morgan PW (1992) Genetic and covered with a cover glass. The slides were then observed with a bright- R field Zeiss microscope. regulation of development in Sorghum bicolor: VIII. ma3 flowering mutant lacks a phytochrome that predominates in green tissue. Plant Physiol 99: 765–770 Bud Length and Seedling Height Measurement Childs KL, Miller FR, Cordonnier-Pratt M-M, Pratt LH, Morgan PW,

Mullet JE (1997) The sorghum photoperiod sensitivity gene, Ma3, Subtending leaves were removed, and buds in the axil of the first leaf were encodes a phytochrome B. Plant Physiol 113: 611–619 excised and their lengths measured under a dissecting microscope using a Cline MG (1997) Concepts and terminology of apical dominance. Am J Bot micrometer. A ruler was used to measure buds longer than 3 mm. Seedling 84: 1064–1069 height was also measured as the height from the base of the shoot to the tip of Cubas P, Lauter N, Doebley J, Coen E (1999) The TCP domain: a motif the tallest leaf. foundinproteinsregulatingplantgrowthanddevelopment.PlantJ18: 215–222 RNA Isolation and Northern Analysis Devlin PF, Yanovsky MJ, Kay SA (2003) A genomic analysis of the shade avoidance response in Arabidopsis. Plant Physiol 133: 1617–1629 For gene expression studies, seedlings were harvested, roots were washed, Doebley J, Stec A (1991) Genetic analysis of the morphological differences and buds in the axil of the first leaves were excised under a dissecting between maize and teosinte. Genetics 129: 285–295 microscope. The buds were immersed in lysis/binding solution (Ambion) on Doebley J, Stec A, Gustus C (1995) teosinte branched1 and the origin of ice and were stored at 280°C until RNA extraction. The roots of seedlings maize: evidence for epistasis and the evolution of dominance. Genetics were kept moist during sampling. Sampling was done between 11 AM to 2 PM 141: 333–346 during the day. Total RNA was extracted using the Trizol method (Life Doebley J, Stec A, Hubbard L (1997) The evolution of apical dominance in Technologies), and then separated on a denaturing glyoxal agarose gel and maize. Nature 386: 385–388 transferred to a Hybond membrane (Amersham Biosciences). Membranes Faivre-Rampant O, Bryan GJ, Roberts AG, Milbourne D, Viola R, Taylor were probed with SbTB1, SbDRM1,andSbUBQ (S. bicolor ubiquitin) genes. MA (2004) Regulated expression of a novel TCP domain transcription Probes were prepared by PCR amplification from a cDNA prepared from factor indicates an involvement in the control of meristem activation sorghum axillary buds using primers for SbTB1, forward 5#-GGTGGTG- processes in Solanum tuberosum. JExpBot55: 951–953 GTTCAAATGGTTC-3# and reverse 5#-TACAATGGCTCCTCAACACG-3#; Foo E, Bullier E, Goussot M, Foucher F, Rameau C, Beveridge CA (2005) for SbDRM1,forward5#-TGGTGGCTTTGTGAGTGAAG-3# and reverse The branching gene RAMOSUS1 mediates interactions among two 5#-TTATCAGCAACAGCGACAGC-3#; and for SbUBQ, forward 5#-GGA- novel signals and auxin in pea. Plant Cell 17: 464–474 AACATAGGGACGCTTCA-3# and reverse AAGGAGTCCACCCTTCACCT-3#. 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